The present invention relates to a novel use of interleukin-7 (IL-7) for treating autoimmune diseases, in particular insulin-dependent diabetes mellitus.
Type 1 diabetes or insulin-dependent diabetes mellitus (IDDM) is nowadays considered to be an autoimmune disease (Endocr. Rev. 1994, 15 (4), 516-542), characterised by the presence of anti-beta cell antibodies and by its sensitivity to immunosuppressive therapy. In both man and in the nonobese diabetic (NOD) mouse, IDDM results from a predominantly cellular type immune response, the humoral response being characterised by secretion of anti-membrane antibodies and secretary beta cell anti-products (N. Engl. J. Med. 1981, 304, 1454-1465, N. Engl. J. Med., 1992, 327, 302). The cellular type immune response is characterised by histological lesions or insulitis caused by infiltration of macrophage and B and T lymphocyte type inflammatory and immune cells into the islets of Langerhans of the pancreas (Diabetologia, 1989, 32, 282-289; Insulitis and type 1 diabetes, Academic Press Tokyo, 1986, 35-50).
The NOD mouse is a spontaneous model of autoimmune diabetes or type 1 diabetes. Converging arguments indicate that the onset of the disease is under the control of CD4+ immunoregulatory T cells. It is accelerated by a thymectomy carried out at 3 weeks of age (Eur. J. Immuno. 1989, 19, 889-895) and can be prevented, in a transfer system, by co-injection of thymocytes or CD4+ splenic cells from young not yet diabetic NOD mice (J. Exp. Med., 1989, 169, 1669-1680).
It has been indirectly suggested and demonstrated that, because of their ability to produce interleukin-4 (IL-4), T helper 2 (Th2) lymphocyte cells are the immunoregulatory CD4+ cells in question (Autoimmunity, 1993, 15, 113-122). It has been demonstrated that administration of IL-4 (J. Exp. Med. 1993, 178 (1), 87-99) or anti-xcex3-interferon (xcex3-IFN) monoclonal antibodies prevent the onset of diabetes and administration of interleukin-12 (IL-12), the T helper 1 (Th1) lymphocyte inducer, accelerates the onset of diabetes (J. Exp. Med., 1995, 181 (2), 817-821).
However, a further study has shown that while diabetologically Th2-like cells discovered infiltrated into the islets do not cause the disease to appear, they do not afford significant protection (Science, 1995, 268 (5214), 1185-1188). Thus while it has been indirectly suggested and shown that the onset of diabetes in the NOD mouse is under the control of Th2 cells, no explanation has been given or suggested regarding the character of the anomaly present in the NOD mouse of the physiological process which occurs which is the origin of the emergence of an anti-Langerhans islets autoimmunity at the origin of diabetes in such animals. It has also recently been suggested that Th2 cell differentiation could be controlled by a T cell sub-type characterised by the TCR-xcex1xcex2, CD4xe2x88x92CD8xe2x88x92 (double negative, DN) or CD4+CD8xe2x88x92 (simple positive) phenotype, carrying a mature phenotype (non-expression of heat stable antigen, HSA), selectively expressing the activation marker CD44. This sub-population, which is also characterised in other strains by the NK1.1 marker, has the ability to produce a large quantity of IL-4 after stimulation with an anti-TCR-xcex1xcex2 polyclonal antibody (TCR-xcex1xcex2: xcex1xcex2 receptor of T cells) (J. Immunol. 1995, 155 (10), 4544-4550).
It has also been shown that this sub-type can be restricted by class I molecules of the major histocompatibility complex (MHC) and preferably uses the Vxcex28 gene for the T receptor (J. Exp. Med., 1993, 178, 901-908), a sub-type proliferation of which is specifically induced by interleukin-7 (J. Exp. Med., 1994, 180 (2), 653-661).
Mammalian interleukin-7 proteins (cytokine IL-7s), in particular in man and in the mouse, the corresponding DNA, expression vectors coding for the IL-7s, and processes for their production, including recombinant systems, have been described (U.S. Pat. No. 4,965,195).
Mammalian interleukin-7 proteins will hereinafter be designated xe2x80x9cIL-7xe2x80x9d. IL-7 is a lymphopoietic growth factor which can stimulate the development and proliferation of bone marrow cells (WO 89/03884). Stimulation of platelet production (WO 90/09194) by induction and proliferation of megacaryocytes was one of the first applications of IL-7. Other IL-7 applications have also been described such as cancer treatment or a treatment of a viral infection by immunotherapy from modified cells producing IL-7, either by direct injection of modified cells in vivo, or by a prior in vitro treatment phase before injection (WO 92/01459). The use of IL-7 for proliferating specific anti-HIV human T lymphocytes as a potential AIDS therapy (J. of Leucocyte Biology, 1995, 58 (6), 623-633), for treating malignant melanoma in dermatology (Hautarzt, 1995, 46 (10), 676-682), and as a potentialiser for a vaccine to prevent infection (microbial and viral) and tumours (WO 94/22473) have also been described. The use of an anti-IL-7 antibody for studying and researching physiological and pathological processes, in particular regarding differentiation and proliferation of lymphocytes (WO 94/28160), has also been described. Other applications combining the use of IL-7 with other cytokines have been described, such as the combination with IL-4 for in vitro induction of pre-B cell differentiation (WO 94/04658) or with IL-3 to treat leucopenia (WO 92/04465) and to prevent bone marrow disorders after cancer therapy or bone marrow grafts (WO 93/03061).
The authors of the present invention have shown here, on the basis of a phenotype study using flow cytometry using HSA, CD4, CD8, CD44 and Vxcex28 markers, that a sub-population of thymocytes with phenotype CD44+TCR-xcex1xcex2 HSAxe2x88x92 and preferably using the Vxcex28 gene of the T receptor is numerically reduced in 3-week old NOD mice (both for the CD4xe2x88x92-CD8xe2x88x92DN (double negative CD4xe2x88x92CD8xe2x88x92) and CD4+ (simple positive) populations and in 8-week old mice (for the DN population). It has also been discovered that at both ages this sub-population produces little or no IL-4. Finally, the authors of the present invention have shown that this double numerical and functional anomaly is corrected by IL-7 in vitro and in vivo, the growth factor for this cellular sub-population, and that IL-7 can protect mammals from diabetes, in particular insulin-dependent diabetes mellitus, this property also possibly extending to all autoimmune diseases, generated by a failure in the production of IL-4 by Th2 cells, in particular by the cellular sub-population described above, and generally to all autoimmune diseases, in particular those generated by a failure in immunoregulation by CD4+ cells.
Thus in a first aspect, the present invention provides for the use of IL-7 or T lymphocytes incubated in the presence of IL-7 or modified lymphocytes producing IL-7 for the preparation of drugs or pharmaceutical compositions for treating autoimmune diseases, in particular autoimmune diseases generated by a failure in immunoregulation by CD4+ T cells.
Preferably, the autoimmune diseases are autoimmune diseases generated by a failure in the production of IL-4 by Th2 cells.
Particularly preferably, the autoimmune diseases are autoimmune diseases generated by a failure in the production of IL-4 connected with a quantitative and functional deficiency of a T cell sub-type with phenotype HSAxe2x88x92, CD4xe2x88x92CD8xe2x88x92 or CD4+CD8xe2x88x92, CD44+, TCR-xcex1xcex2+, Vxcex28+, NK1.1+.
In addition to insulin-dependent diabetes mellitus, other autoimmune diseases, autoimmune encephalo-myelitis, autoimmune rheumatoid arthritis, polyarthritis, autoimmune type 2 hepatitis, autoimmune gastritis, autoimmune sclerosis, sialadenitis, adrenalitis, oophoritis, glomerulonephritis, and autoimmune thyroiditis, can preferably be treated by said drugs or compositions, as can any autoimmune type pathogenic mechanism in a therapy associated with treatment of AIDS.
The autoimmune disease is preferably insulin-dependent diabetes mellitus.
Advantageously, the T lymphocytes previously incubated in the presence. of IL-7, used in the present invention, are autological or syngeneic cells from cells of the patients for whom the compositions comprising them are intended.
The invention also encompasses pharmaceutical compositions offering a novel approach for treating autoimmune diseases generated by a failure in immunoregulation by CD4+ T cells.
In particular, the invention encompasses pharmaceutical compositions offering a novel approach for treating autoimmune diseases generated by a failure in the production of IL-4 by Th2 cells, particularly autoimmune diseases generated by a failure in IL-4 production connected with a quantitative and functional deficiency of a T cell sub-type with phenotype HSAxe2x88x92, CD4xe2x88x92CD8xe2x88x92 or CD4+CD8xe2x88x92, CD44+, TCR-xcex1xcex2+, Vxcex28+, NK1.1+, in particular insulin-dependent diabetes mellitus.
Such compositions comprise IL-7 as the active principle, preferably in its soluble form, and/or autologous or syngeneic T lymphocytes from cells of a patient for whom the pharmaceutical composition is intended, said T lymphocytes having previously been incubated in the presence of IL-7. They can also be in the form of combinations with other active principles, for example other immunomodulating agents.
These different compositions can be administered in a number of different ways, as the skilled person will be able to determine, depending on the type of composition concerned.
Compositions comprising IL-7 as the active principle can be administered systemically, for example, preferably intravenously, intramuscularly, intradermally or orally.
Compositions comprising T lymphocytes as the active principle are preferably administered intravenously or intraperitoneally.
Preferred modes of administration, also dosages and optimum galenical forms, can be determined using the criteria which are generally considered in establishing a therapeutic treatment which is tailored to a patient, for example age or body weight of the patient, the seriousness of their general condition, tolerance to treatment and any known side effects.
The present invention also relates to a process for the production of a drug or pharmaceutical composition for treating autoimmune diseases, in particular autoimmune diseases generated by a failure in immunoregulation by CD4+ T cells, characterised in that IL-7 and/or autologous or syngeneic T lymphocytes from cells of the patient for whom the composition is intended are mixed, said T lymphocytes having previously been incubated in the presence of IL-7 with a pharmaceutically acceptable vehicle or diluent, optionally combined with other active principles.
In particular, the invention concerns a process for the production of a drug or pharmaceutical composition for treating autoimmune diseases generated by a failure in IL-4 production by Th2 cells, in particular autoimmune diseases generated by a failure in IL-4 production connected with a quantitative and functional deficiency of a T cell sub-type with phenotype HSAxe2x88x92, CD4xe2x88x92CD8xe2x88x92 or CD4+CD8xe2x88x92, CD44+, TCR-xcex1xcex2+, Vxcex28+, NK1.1+, in particular insulin-dependent diabetes mellitus.
The present invention concerns a therapeutic treatment method characterised in that a therapeutically effective dose of IL-7 or T lymphocytes which have previously been incubated in the presence of IL-7 is administered to a patient with an autoimmune disease, in particular an autoimmune disease generated by a failure in immunoregulation by CD4+ cells.
Advantageously, the method of the invention is applicable to treating insulin-dependent diabetes mellitus.
The present invention is illustrated by the accompanying FIGS. 1 and 2.
FIG. 1 shows the in vitro correcting effect of IL-7 on IL-4 production by mature HSAxe2x88x92CD8xe2x88x92 thymocyte cells in the young (FIG. 1a) and in the adult (FIG. 1b) NOD and C57BL/6 mouse.
In the experiment shown, freshly harvested HSAxe2x88x92CD8xe2x88x92 thymocytes from young (3 weeks old) or adult (8 weeks old) NOD or C57BL/6 mice were cultivated to 1.0xc3x97105 cells/well with an anti-TCR-xcex1xcex2 monoclonal antibody in the presence (hatched column) or absence (open column) of 1000 U/ml of IL-7. The supernatant was recovered after 48 hours of incubation and measured for IL-4 using ELISA (average of 3 or 4 independent experiments).
Other features and advantages of the invention are apparent from the remaining description including the Examples and Tables summarising the results of the experiments.
FIG. 2 shows the effect of treating mice with IL-7 on the primary IL-4 production by splenocytes.
In the experiment shown, mice of different strains aged from 8 to 12 weeks received daily sub-cutaneous injections of 2 xcexcg of IL-7 or bovine serum albumin (excipient) over 7 consecutive days. On the eight day, the mice were treated with anti-CD3 antibody then the cultivated splenocytes (5xc3x97106 cells per well) and IL-4 production was measured using the CT.4S test. The percentage increase in IL-4 production was calculated as follows: ((quantity of IL-4 produced after treatment with IL-7)/(quantity of IL-4 produced after treatment with excipient)xe2x88x921)xc3x97100. The results are those of a typical experiment. The production of IL-4 (U/ml) by mouse splenocytes treated with excipient was respectively 94.3; 53.5; 11.0; and 11.4 for BALB/c, C57BL/6, NOD and C57BL/6xcex22mxe2x88x92/xe2x88x92.